Methods for treating diabetes

ABSTRACT

Methods for treating diabetes by administering an inhibitor of GDF-8, or a related member of Transforming Growth Factor-beta (TGF-β) superfamily of structurally-related growth factors (e.g., GDF-11) are disclosed. Also disclosed are methods for upregulating expression of hexose transporters, such as GLUT4 and GLUT1, in a subject by administering an inhibitor of GDF-8. Also disclosed are methods for increasing glucose uptake by cells in a subject, by administering an inhibitor of GDF-8.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/084,490, filed May 6, 1998, and is a continutation of U.S.application Ser. No. 09/988,835, filed Nov. 19, 2001 which is acontinuation of U.S. application Ser. No. 09/305,989, filed May 6, 1999the entire contents of which are hereby incorporated by referenceherein.

BACKGROUND OF THE INVENTION

Diabetes mellitus is the most common metabolic disease worldwide. Everyday, 1700 new cases of diabetes are diagnosed in the United States, andat least one-third of the 16 million Americans with diabetes are unawareof it. Diabetes is the leading cause of blindness, renal failure, andlower limb amputations in adults and is a major risk factor forcardiovascular disease and stroke.

Normal glucose homeostasis requires the finely tuned orchestration ofinsulin secretion by pancreatic beta cells in response to subtle changesin blood glucose levels, delicately balanced with secretion ofcounter-regulatory hormones such as glucagon. Type 1 diabetes resultsfrom autoimmune destruction of pancreatic beta cells causing insulindeficiency. Type 2 or noninsulin-dependent diabetes mellitus (NIDDM)accounts for >90% of cases and is characterized by a triad of (1)resistance to insulin action on glucose uptake in peripheral tissues,especially skeletal muscle and adipocytes, (2) impaired insulin actionto inhibit hepatic glucose production, and (3) dysregulated insulinsecretion (DeFronzo, (1997) Diabetes Rev. 5:177-269). In most cases,type 2 diabetes is a polygenic disease with complex inheritance patterns(reviewed in Kahn et al., (1996) Annu. Rev. Med. 47:509-531).

Environmental factors, especially diet, physical activity, and age,interact with genetic predisposition to affect disease prevalence.Susceptibility to both insulin resistance and insulin secretory defectsappears to be genetically determined (Kahn, et al). Defects in insulinaction precede the overt disease and are seen in nondiabetic relativesof diabetic subjects. In spite of intense investigation, the genesresponsible for the common forms of Type 2 diabetes remain unknown.

One of the fundamental actions of insulin is to stimulate uptake ofglucose from the blood into tissues, especially muscle and fat. Thisoccurs via facilitated diffusion which is mediated by specific glucosetransporter proteins that insert into the plasma membrane of cells.GLUT4 is the most important insulin-sensitive glucose transporter inthese tissues. Insulin binds to its receptor in the plasma membrane,generating a series of signals that result in the translocation ormovement of GLUT4 transporter vesicles to the plasma membrane, where afirst docking step, followed by fusion with the plasma membrane takesplace; after an activation or exposure step takes place, glucose entersthe cell. Studies in both animals and humans indicate that alterationsin GLUT4 expression, trafficking, and/or activity occur in adipose cellsand muscle in diabetes and other insulin-resistant states (Abel et al.,Diabetes Mellitus: A Fundamental and Clinical Text (1996) pp.530-543.)

New and innovative treatments for diabetes are clearly a priority forresearchers in this field. The present invention provides suchinnovative treatments, taking advantage of the knowledge concerningGLUT4 expression and activity, and expression and activity of relatedhexose transporters (e.g., GLUT1).

SUMMARY OF THE INVENTION

The present invention provides a method of treating diabetes and relateddiseases, such as obesity, by administering to a subject an inhibitor ofGDF-8. Suitable inhibitors of GDF-8 which can be employed in the methodsof the invention include, but are not limited to, GDF-8 peptides (e.g.,derived from the pro-domain), GDF-8 dominant-negative mutants,antibodies and antibody fragments which bind to GDF-8 (or the receptorfor GDF-8) and inhibit GDF-8 binding to its receptor, GDF-8 receptorpeptide antagonisists, antisense nucleic acids directed against GDF-8mRNA and anti-GDF-8 ribozymes.

In another aspect, the present invention provides a method of increasingGLUT4 expression in a cell (e.g., a muscle cell or a fat cell in asubject), or increasing glucose uptake by a cell, by administering aGDF-8 inhibitor. Such methods can be used, not only to treat diabetesand related diseases, but also to treat several systemic problemsresulting from insufficient glucose metabolism, such as hyperglycemia.

The methods of the present invention also can be performed using astargets other TGF-β growth factors which are related in structure andactivity to GDF-8, such as GDF-11. Accordingly, in another embodiment,the invention provides method of treating diabetes by administering to asubject an inhibitor of GDF-11, either alone or incombination with otherGDF inhibitors (e.g., an inhibitor of GDF-8).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows GLUT4 levels by immunostaining, with an anti-GLUT4antibody, in the pectoralis and the quadriceps from a wild-type mouseand a GDF-8 knockout mouse.

FIG. 1B shows GLUT4 levels, by immunostaining, with an anti-GLUT4antibody, in five different muscle samples, pectoralis, triceps,gastrocnemius, quadriceps, and iliocostal, in both a wild-type mouse anda GDF-8 knockout mouse.

FIG. 2 shows GLUT4 levels by immunostaining with an anti-GLUT4 antibodyin muscle from a control mouse, a GDF-8-dosed mouse, an insulin-dosedmouse, and a GDF-8 plus insulin-dosed mouse.

FIG. 3 is a graph showing the correlation between increased systemiclevels of GDF-8 in nude mice (as secreted from a GDF-8-expressing CHOcell tumor) and severe weight loss as compared to control mice.

FIG. 4 is a graph showing the correlation between increased systemiclevels of GDF-8 in nude mice (as secreted from a GDF-8-expressing CHOcell tumor) and overall body weight (Panel A), tumor weight (Panel B),pectoralis weight (Panel C), epididymal fat weight (Panel D) andgastrocnemius weight (Panel E) as compared to these tissues from controlmice containing CHO cell tumors not expressing GDF-8.

FIG. 5 is a graph comparing the size of GDF-8-secreting CHO cell tumorsin nude mice relative to control CHO cell tumors not expressing GDF-8.Tumor size was measured as cross sectional area.

FIG. 6 is a graph showing the correlation between increased GDF-8 levels(from GDF-8 expressing CHO cell tumors) in nude mice and serum glucoselevels (Panel A) and GLUT4 expression levels (Panel B) in muscle, ascompared to control mice containing CHO cell tumors not expressingGDF-8.

FIG. 7 shows the effect of exogenously added GDF-8 on 3T3-L1 adipocytedifferentiation, as compared to no treatment, TNF-α treatment, andTGF-β1 treatment.

FIG. 8 is a Northern blot analysis showing reduced expression of GLUT4in 3T3-L1 cells treated with GDF-8, TGFβ and TNFα. Total cellular RNAfor each sample was fractionated, immobilized to a membrane andhybridized with ³²P probes for GLUT4 mRNA.

FIG. 9 is a graph showing that treatment of differentiated 3T3-L1adipocytes with different doses of GDF-8 impairs the ability of thesecells to increase glucose uptake in response to insulin, thus leading todesensitization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that GDF-8downregulates expression of GLUT4 in tissues primarily in muscle andfat. Regulation of glucose metabolism by insulin is a key mechanism bywhich homeostasis is maintained in an animal. The action of insulin inthe regulation of circulating glucose levels is to stimulate glucoseuptake in muscle and fat tissues. Insulin stimulates glucose uptake inthese tissues by increasing the translocation of GLUT4, theinsulin-sensitive glucose transporter, from an intracellular vesicularcompartment to the plasma membrane.

It was further discovered as part of the present invention that GLUT4expression in muscle and fat cells can be upregulated by inhibitingGDF-8. It was also discovered that glucose uptake by these cells can beincreased by inhibiting GDF-8. These effects can be advantageouslyutilized to treat a variety of metabolic diseases resulting fromdysfunctional glucose metabolism (e.g., hyperglycemia) and/or insulinresistance.

Accordingly, in one embodiment, the present invention provides a methodfor treating diabetes mellitus and related disorders, such as obesity orhyperglycemia, by administering to a subject an inhibitor of GDF-8 in anamount sufficient to ameliorate the symptoms of the disease. Type 2 ornoninsulin-dependent diabetes mellitus (NIDDM), in particular, ischaracterized by a triad of (1) resistance to insulin action on glucoseuptake in peripheral tissues, especially skeletal muscle and adipocytes,(2) impaired insulin action to inhibit hepatic glucose production, and(3) dysregulated insulin secretion (DeFronzo, (1997) Diabetes Rev.5:177-269). Therefore, subjects suffering from type 2 diabetes can betreated according to the present invention by administration of a GDF-8inhibitor, which increases sensitivity to insulin and glucose uptake bycells.

Similarly, other diseases characterized by insulin dysfunction (e.g.,resistance, inactivity or deficiency) and/or insufficient glucosetransport into cells also can be treated according to the presentinvention by administration of a GDF-8 inhibitor, which increasessensitivity to insulin and glucose uptake by cells.

Definitions

As used herein, the term “GDF-8 inhibitor” or “an inhibitor of GDF-8”includes any agent capable of inhibiting GDF-8 activity, including butnot limited to peptides (derived from GDF-8, GDF-11 or other unrelatedsequences), dominant-negative protein mutants, peptidomimetics,antibodies or fragments thereof, ribozymes, antisense oligonucleotides,or other small molecules which specifically inhibit the action of GDF-8while, preferably, leaving intact the activity of TGF-β, Activin orother members of the TGF-β superfamily. The term “a GDF-11 inhibtor”also encompasses these classes of inhibitors and, preferably,specifically inhibits GDF-11. GDF-8 and GDF-11 are structurally andfunctionally related members of the TGF-β family of growth factors.

GDF-8 inhibitors used in the methods of the invention, particularlythose derived from GDF-8 itself (e.g., GDF-8 peptides, such as thepro-domain or portions thereof), preferably do not possess GDF-8activity. Such inhibitors and methods for their identification aredescribed in U.S. Ser. No. 60/116,639, entitled “Growth DifferentiationFactor Inhibitors and Uses Therefor”, incorporated by reference hereinin its entirety. For example, the inhibitory action of a GDF-8 inhibitorcan be assessed using a variety of art-recognized assays, such as aNorthern blot analysis of GDF-8 mRNA, or a Western blot analysis orimmunostaining analysis of GDF-8 protein levels, among others.Identified GDF-8 inhibitory compounds can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki et al., Bio/Technology, 3:1008 (1985)), allele-specificoligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad.Sci. USA 80:278 (1983)), ligase-mediated gene detection (Landegren etal, Science 241:1077 (1988)), and the like.

As used herein, the term “GDF-8 activity” or “GDF-11 activity” includesany activity mediated by GDF-8 or GDF-11, respectively. For example,GDF-8 is known to inhibit fibroblast differentiation to adipocytes,modulate the production of muscle-specific enzymes, e.g., creatinekinase, modulate uptake glucose by cells, and stimulate myoblast cellproliferation. Accordingly, the degree to which a GDF-8 inhibitorprevents GDF-8 activity can be identified by, for example, testing forthe ability of the inhibitor to block GDF-8 activity, as measured by theability of GDF-8 to interfere with the differentiation process of 3T3-L1pre-adipocytes (fibroblasts) to adipocytes, the ability to modulate theactivity of muscle-specific enzymes, e.g., creatine kinase, the abilityto modulate glucose uptake by cells, or the ability to stimulatemyoblast cell proliferation. The effect of the inhibitor on inhibitionof insulin stimulation of GLUT4 expression and glucose uptake can alsobe assessed, and may include measurements before and after incubating inthe presence of the compound.

As used herein, the term “modulate” refers to an increase in function.For example, modulation of gene transcription or expression refers toupregulation of these functions. Modulation of protein activity refersto an increase in activity.

As used herein, the term “inhibit” refers to a decrease, whether partialor whole, in function. For example, inhibition of gene transcription orexpression refers to any level of downregulation of these functions,including complete elimination of these functions. Modulation of proteinactivity refers to any decrease in activity, including completeelimination of activity.

As used herein, the term “diabetes” includes all known forms ofdiabetes, including type I and type II diabetes, as described in Abel etal., Diabetes Mellitus: A Fundamental and Clinical Text (1996)pp.530-543.

GDF-8 inhibitors of the invention are typically administered to asubject in “substantially pure” form. The term “substantially pure” asused herein refers to GDF-8 which is substantially free of otherproteins, lipids, carbohydrates, or other materials with which it isnaturally associated. One skilled in the art can purify GDF-8 usingstandard techniques for protein purification. The substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel. The purity of the GDF-8 polypeptide can also bedetermined by amino-terminal amino acid sequence analysis.

Specific details on the production of GDF-8 for testing and developinginhibitors for use in the present invention are provided by McPherron,et al., Nature 387:83 90 (1997), and U.S. Pat. No. 5,827,733, bothhereby incorporated by reference in their entirety.

As used herein, the term “hexose transporter” includes integral membraneproteins of a cell able to transport a hexose sugar, such as glucose,from the exterior to the interior of the cell. Examples of suchtransporters are the GLUT1 and GLUT4 transporter proteins, among others,in muscle and fat cells.

As used herein, the term “modulation of GDF-8 activity” or “modulationof GDF-8 level” refers to a change in GDF-8 activity or level comparedto its native state. This change may be either positive (upregulation),or negative (downregulation), but for the purposes of the presentinvention is preferably the latter.

Cells which are targeted by the methods of the present invention, suchas muscle and fat cells, include isolated cells maintained in culture aswell as cells within their natural context in vivo (e.g., in fat tissueor muscle tissue, such as pectoralis, triceps, gastrocnemius,quadriceps, and iliocostal muscles).

The term “antisense nucleic acid” refers to a DNA or RNA molecule thatis complementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American 262:40 (1990)). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate an mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetGDF-8 producing cell. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal. Biochem. 172:289 (1988)).

As used herein, a “ribozyme” is a nucleic acid molecule having nucleaseactivity for a specific nucleic acid sequence. A ribozyme specific forGDF-8 mRNA, for example, would bind to and cleave specific regions ofthe GDF-8 mRNA, thereby rendering it untranslatable and resulting inlack of GDF-8 polypeptide production.

The term “dominant-negative mutant” refers to a GDF-8 protein which hasbeen mutated from its natural state and which interacts with GDF-8 or aGDF-8 gene, thereby inhibiting its production and/or activity.

The “antibodies” of the present invention include antibodiesimmunoreactive with GDF-8 polypeptides or finctional fragments thereof.Antibodies which consist essentially of pooled monoclonal antibodieswith different epitopic specificities, as well as distinct monoclonalantibody preparations are provided. Monoclonal antibodies are made fromantigen-containing fragments of the protein by methods well known tothose skilled in the art (Kohler et al, Nature 256:495(1975)). The term“antibody” as used in this invention is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂, Fv andSCA fragments which are capable of binding an epitopic determinant onGDF-8.

A “Fab fragment” consists of a monovalent antigen-binding fragment of anantibody molecule, and can be produced by digestion of a whole antibodymolecule with the enzyme papain, to yield a fragment consisting of anintact light chain and a portion of a heavy chain.

A “Fab′ fragment” of an antibody molecule can be obtained by treating awhole antibody molecule with pepsin, followed by reduction, to yield amolecule consisting of an intact light chain and a portion of a heavychain. Two Fab′ fragments are obtained per antibody molecule treated inthis manner.

A “(Fab′)₂” of an antibody can be obtained by treating a whole antibodymolecule with the enzyme pepsin, without subsequent reduction. A (Fab′)₂fragment is a dimer of two Fab′ fragments held together by two disulfidebonds.

An “Fv fragment” is defined as a genetically engineered fragmentcontaining the variable region of a light chain and the variable regionof a heavy chain expressed as two chains.

A “single chain antibody” (SCA) is a genetically engineered single chainmolecule containing the variable region of a light chain and thevariable region of a heavy chain, linked by a suitable, flexiblepolypeptide linker.

GDF-8 and GDF-11 Inhibitors for use in the Methods of the Invention

GDF-8 inhibitors suitable for use in the invention include, but are notlimited to, peptides, including peptides derived from GDF-8 (e.g.,mature GDF-8 or the pro-domain of GDF-8) or non-GDF-8 peptides, GDF-8dominant-negative mutants, antibodies and antibody fragments which bindto GDF-8 (or the receptor for GDF-8) and inhibit GDF-8 binding to itsreceptor, GDF-8 receptor peptide antagonisists, antisense nucleic acidsdirected against GDF-8 mRNA and anti-GDF-8 ribozymes. Thus, GDF-8inhibitors can act at the message (transcription) level or at theprotein (expression or activity) level.

As used herein, the term “GDF-8” includes all known forms of GDF-8including but not limited to human GDF-8, bovine GDF-8, chicken GDF-8,murine GDF-8, rat GDF-8, porcine GDF-8, ovine GDF-8, turkey GDF-8, andbaboon GDF-8. These molecules are described in McPherron A. C. et al.(1997) Proc. Natl. Acad. Sci. 94:12457-12461, the contents of which areincorporated herein by reference. The amino acid sequences for theseproteins are shown in FIG. 12.

As used herein, the term “GDF-11” includes all known forms of GDF-11including but not limited to human GDF-11, bovine GDF-11, chickenGDF-11, murine GDF-11, rat GDF-11, porcine GDF-11, ovine GDF-11, turkeyGDF-11, and baboon GDF-11.

GDF-8 and GDF-11 inhibitory peptides can be identified and isolated frommedia of cells expressing GDF-8 or GDF-11 using techniques known in theart for purifying peptides or proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for the GDF-8 or GDF-11 inhibitor, or a portion thereof. In oneembodiment, the media obtained from cultures of cells which expressGDF-8 or GDF-11 are subjected to high performance liquid chromatography(HPLC). The samples obtained can then be tested for GDF-8 or GDF-11inhibitory activity as described below.

Alternatively, GDF-8 and GDF-11 peptide inhibitors can be identified byscreening fragments of GDF-8 or GDF-11 for inhibitory activity. GDF-8 orGDF-11 fragments can be produced by a variety of art known techniques.For example, specific oligopeptides (approximately 10-25 aminoacids-long) spanning the GDF-8 or GDF-11 sequence can be synthesized(e.g., chemically or recombinantly) and tested for their ability toinhibit GDF-8 or GDF-11, for example, using the assays described herein.The GDF-8 or GDF-11 peptide fragments can be synthesized using standardtechniques such as those described in Bodansky, M. Principles of peptideSynthesis, Springer Verlag, Berlin (1993) and Grant, G. A (ed.).Synthetic Peptides: A User's Guide, W.H. Freeman and Company, New York(1992). Automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600).

Alternatively, GDF-8 or GDF-11 fragments can be produced by digestion ofnative or recombinantly produced GDF-8 or GDF-11 by, for example, usinga protease, e.g., trypsin, thermolysin, chymotrypsin, or pepsin.Computer analysis (using commercially available software, e.g.MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used toidentify proteolytic cleavage sites.

GDF-8 or GDF-11 inhibitors used in the methods of the invention arepreferably isolated. As used herein, an “isolated” or “purified” proteinor biologically active peptide thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the GDF-8 or GDF-11 protein or peptide is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of GDF-8 or GDF-11 protein or peptidethereof in which the protein or peptide thereof is separated fromcellular components of the cells from which it is isolated orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of GDF-8 or GDF-11protein or peptide thereof having less than about 30% (by dry weight) ofnon-GDF-8 or GDF-11 protein or peptide thereof (also referred to hereinas a “contaminating protein”), more preferably less than about 20% ofnon-GDF-8 or GDF-11 protein or peptide thereof, still more preferablyless than about 10% of non-GDF-8 or GDF-11 protein or peptide thereof,and most preferably less than about 5% non-GDF-8 or GDF-11 protein orpeptide thereof. When the GDF-8 or GDF-11 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

A two-step method can be used to produce and isolate suchproteolytically cleaved GDF-8 or GDF-11 peptides. The first stepinvolves enzymatic digestion of the GDF-8 or GDF-11 protein. GDF-8 orGDF-11 can be produced either as a dimer from CHO cell conditionedmedia, as a monomer in E. coli or yeast, or isolated from cells whichnaturally produce GDF-8 or GDF-11. Following purification of GDF-8 orGDF-11 monomers or dimers by, for example, HPLC chromatography, theirenzymatic digestion is performed as described infra. The amino acidscleaved during the digestion depend on the specific protease used in theexperiment as is known in the art. For example, if the protease ofchoice were trypsin, the cleavage sites would be amino acids arginineand lysine. The GDF-8 or GDF-11 protein can be digested using one ormore of such proteases.

After the digestion, the second step involves the isolation of peptidefractions generated by the protein digestion. This can be accomplishedby, for example, high resolution peptide separation as described infra.Once the fractions have been isolated, their GDF-8 or GDF-11 inhibitoryactivity can be tested for by an appropriate bioassay, as describedbelow.

The proteolytic or synthetic GDF-8 or GDF-11 fragments can comprise asmany amino acid residues as are necessary to inhibit, e.g., partially orcompletely, GDF-8 or GDF-11 function, and preferably comprise at least5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100 or more amino acids in length.

In one embodiment, peptides are selected which do not contain asufficient number of T cell epitopes to induce T cell mediated immuneresponses and/or which contain a sufficient number of B cell epitopes toelicit antibodies when administered to a mammal. Preferred GDF-8 orGDF-11 peptide inhibitors do not contain a sufficient number of T cellepitopes to induce T-cell mediated (e.g., cytokine) responses. However,B cell epitopes may be desirable and can be selected for by, forexample, testing the peptide's ability to elicit an antibody response,as discussed below.

T cell epitopes within GDF-8 or GDF-11 fragments can be identified usinga number of well known techniques. For example, T cell epitopes can bepredicted using algorithms (see e.g., Rothbard, J. and Taylor, W. R.(1988) EMBO J. 7:93-100; Berzofsky, J. A. (1989) Philos Trans R. Soc.Lond 323:535-544). Preferably, human T cell epitopes within a GDF-8 orGDF-11 protein can be predicted using known HLA class II bindingspecific amino acid residues. One algorithm for predicting peptideshaving T cell stimulating activity which has been used with success isreported in Rothbard, 1st Forum in Virology, Annals of the PasteurInstitute, pp 518-526 (December, 1986), Rothbard and Taylor, (1988)Embo, 7:93-100 and EP 0 304 279. These documents report defining ageneral T cell pattern (algorithm), its statistical significance and itscorrelation with known epitopes as well as its successful use inpredicting previously unidentified T cell epitopes of various proteinantigens and autoantigens. The general pattern for a T cell epitope asreported in the above-mentioned documents appears to contain a linearpatter composed of a charged amino acid residue or glycine followed bytwo hydrophobic residues. Other algorithms that have been used topredict T cell epitopes of previously undefined proteins include analgorithm reported by Margalit et al., (1987) J. Immunol.,138:2213-2229, which is based on an amphipathic helix model.

Other methods for identifying T cell epitopes involve screening GDF-8 orGDF-11 inhibitory peptides of the invention for human T cell stimulatingactivity. This can be accomplished using one or more of severaldifferent assays. For example, in vitro, T cell stimulatory activity canbe assayed by contacting a peptide of the invention with an antigenpresenting cell which presents appropriate MHC molecules in a T cellculture. Presentation of a GDF-8 or GDF-11 inhibitory peptide of theinvention in association with appropriate MHC molecules to T cells, inconjunction with the necessary costimulation can have the effect oftransmitting a signal to the T cell that induces the production ofincreased levels of cytokines, particularly of interleukin-2 andinterleukin-4. The culture supernatant can be obtained and assayed forinterleukin-2 or other known cytokines. For example, any one of severalconventional assays for interleukin-2 can be employed, such as the assaydescribed in Proc. Natl. Acad Sci USA, 86:1333 (1989) the entirecontents of which are incorporated herein by reference. A kit for anassay for the production of interferon is also available from GenzymeCorporation (Cambridge, Mass.).

A common assay for T cell proliferation entails measuring tritiatedthymidine incorporation. The proliferation of T cells can be measured invitro by determining the amount of ³H-labeled thymidine incorporatedinto the replicating DNA of cultured cells. Therefore, the rate of DNAsynthesis and, in turn, the rate of cell division can be quantified.

Other preferred peptide inhibitors of GDF-8 or GDF-11 are located on thesurface of the GDF-8 and GDF-11 proteins, e.g., hydrophilic regions, aswell as regions with high antigenicity or fragments with high surfaceprobability scores can be identified using computer analysis programswell known to those of skill in the art (Hopp and Wood, (1983), Mol.Immunol., 20, 483-9, Kyte and Doolittle, (1982), J. Mol. Biol., 157, 105-32, Corrigan and Huang, (1982), Comput. Programs Biomed, 3, 163-8).

Still other preferred peptides of GDF-8 or GDF-11 to be tested for GDF-8or GDF-11 inhibitory activity include one or more B-cell epitopes. Suchpeptides can be identified by immunizing a mammal with the peptide,either alone or combined with or linked to an adjuvant (e.g., a hapten),and testing sera from the immunized animal for anti-GDF-8 or GDF-11antibodies. Preferred peptides generate anti-GDF-8 or GDF-11 antibodieswhich inhibit GDF-8 or GDF-11 activity, indicating that these peptidesare somehow related to the protein's activity (e.g., correspond to allor a portion of the active site). For example, sera from immunizedanimals can be tested for GDF-8 or GDF-11 inhibitory activity using anyof the GDF-8 or GDF-11 bioassays described herein.

Alternatively, anti-GDF-8 or anti-GDF-11 antibodies or antibodyfragments can be administered directly to a subject to inhibit GDF-8 orGDF-11 activity. Preferred antibodies include monoclonal antibodies,including humanized, chimeric and human monoclonals or fragmentsthereof.

To generate such antibodies, a proteolytic or synthetic GDF-8 or GDF-11fragment (alone or linked to a suitable carrier or hapten) can be usedto immunize a subject (e.g., a mammal including, but not limited to arabbit, goat, mouse or other mammal). For example, the methods describedin U.S. Pat. Nos. 5,422,110; 5,837,268; 5,708,155; 5,723,129; and5,849,531, can be used and are incorporated herein by reference. In apreferred embodiment, the mammal being immunized does not containendogenous GDF-8 or GDF-11 (e.g., a GDF-8 or GDF-11 knock-out transgenicanimal). The immunogenic preparation can further include an adjuvant,such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic proteolytic or synthetic GDF-8 or GDF-11 fragmentpreparation induces a polyclonal anti-GDF-8 or GDF-11 antibody response.The anti-GDF-8 or GDF-11 antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized GDF-8 or GDF-11.Subsequently, the sera from the immunized subjects can be tested fortheir GDF-8 or GDF-11 inhibitory activity using any of the bioassaysdescribed herein.

Alternatively, is also possible to immunize subjects (e.g., GDF-8 andGDF-11 knockout mice) with plasmids expressing GDF-8 and GDF-11 usingDNA immunization technology, such as that disclosed in U.S. Pat. No.5,795,872, Ricigliano et al., “DNA construct for immunization” (1998),and in U.S. Pat. No. 5,643,578, Robinson et al., “Immunization byinoculation of DNA transcription unit” (1997).

The antibody molecules directed against GDF-8 or GDF-11 can be isolatedfrom the mammal (e.g., from the blood) and further purified by wellknown techniques, such as protein A chromatography to obtain the IgGfraction. At an appropriate time after immunization, e.g., when theanti-GDF-8 or GDF-11 antibody titers are highest, antibody-producingcells can be obtained from the subject and used to prepare e.g.,monoclonal antibodies by standard techniques, such as the hybridomatechnique originally described by Kohler and Milstein (1975) Nature256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46;Brown et al. (1980) J. Biol. Chem 255:4980-83; Yeh et al. (1976) Proc.Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer29:269-75), the more recent human B cell hybridoma technique (Kozbor etal. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing monoclonalantibody hybridomas is well known (see generally R. H. Kenneth, inMonoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J.Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet.3:231-36). Briefly, an immortal cell line (typically a myeloma) is fusedto lymphocytes (typically splenocytes) from a mammal immunized with aGDF-8 or GDF-11 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds GDF-8 or GDF-11.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-GDF-8 or GDF-11 monoclonal antibody (see, e.g., G. Galfre et al.(1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindGDF-8 or GDF-11, e.g., using a standard ELISA assay. The antibodies canthen be tested for GDF-8 or GDF-11 inhibitory activity using, forexample, the assays described herein.

In another aspect of the invention, GDF-8 protein fragments comprise allor a portion of the GDF-8 pro-domain. The pro-domain of TGF-β has beenshown to have inhibitory activity against the mature active TGF-β(Bottinger et. al., (1996) PNAS, 93, 5877-5882; Gentry and Nash, (1990)Biochemistry, 29, 6851-6857). Since GDF-8 is a member of the TGF-βsuperfamily, the pro-domain of GDF-8 may also act as an inhibitor to theactive GDF-8. The pro-domain of GDF-8 can be generated by expressing itusing various expression systems (e.g. CHO, baculovirus and the like).The expressed pro-domain of GDF-8 can be purified by, for example, usingthe method described in Bottinger et. al. (supra) or any other artrecognized method for purifying peptides. Alternatively, the pro-domaincan be tagged to, for example, FLAG or 6-His, as described below.

Based on the information obtained for TGF-β, peptide fragments that spanthe C-terminus of the mature GDF-8 can be designed and synthesized.Preferably, the GDF-8 peptide fragments are about 25 amino acids long.In other preferred embodiments, the GDF-8 peptide fragments can have asequence length of about, 20-25, 25-30, 30-35, 35-40, or 40-45 aminoacid residues in length. The GDF-8 peptide fragments modeled after theaforementioned pentacosapeptide can then be tested for GDF-8 or GDF-11inhibitory activity using the assays described herein.

GDF-8 or GDF-11 inhibitors for use in the methods of the presentinvention can be identified using a variety of appropriate bioassayswhich test for the inhibition of GDF-8 or GDF-11 activity. The abilityof the GDF-8 or GDF-11 inhibitors to inhibit GDF-8 or GDF-11 activity ispreferably specific, i.e., the GDF-8 inhibitor can specifically inhibitthe GDF-8 protein and the GDF-11 inhibitor can specifically inhibit theGDF-11 protein. In certain embodiments, the GDF-8 inhibitor is also ableto inhibit GDF-11 activity and the GDF-11 inhibitor is also able toinhibit GDF-8 activity.

As used herein, the term “bioassay” includes any assay designed toidentify a GDF-8 or GDF-11 inhibitor. The assay can be an in vitro or anin vivo assay suitable for identifying whether a GDF-8 or GDF-11inhibitor can inhibit one or more of the biological functions of GDF-8or GDF-11. Examples of suitable bioassays include DNA replicationassays, transcription-based assays, creatine kinase assays, assays basedon the differentiation of 3T3-L1 pre-adipocytes, assays based on glucoseuptake control in 3T3-L1 adipocytes, and immunological assays (describedin subsection II).

It has been established that GDF-8 modulates the protein levels, andtherefore the activity, of a muscle-specific enzyme, creatine kinase.This effect of GDF-8 or GDF-11 can be used to screen fractions thatcontain potential GDF-8 or GDF-11 inhibitors. This assay can beperformed in the mouse skeletal myoblast cell line C1C12 or in primarychick myoblasts isolated from Day11 chick embryos. Cells are grown in48-well trays in serum-containing medium that maintains themundifferentiated. When a 70% confluence has been reached, medium isswitched to 1% serum, thus allowing differentiation and creatine kinaseexpression. At the time of the switch, the potential GDF-8 orGDF-11-inhibitory fraction is added to some wells, followed some timelater by GDF-8 or GDF-11 itself. Cells are returned to the incubator foran additional two to three day period. In the end, cells are lysed andcreatine kinase activity is measured in the lysates using a commerciallyavailable kit (available by Sigma, St Louis, Mo.).

Uses

In one embodiment, the method of the invention can be used either invitro or in vivo to modulate (i.e., upregulate) the expression of ahexose transporter, such as GLUT4 or GLUT1, in a cell which expressesthese transporters, such as a muscle and/or fat cell. This is achievedby inhibiting the activity or expression of GDF-8 or GDF-11 in the cellor outside the cell.

In another embodiment, the method of the invention can be used either invitro or in vivo to increase insulin sensitivity and/or glucose uptakeby a cell.

In another embodiment, the method of the invention can be used to treata disease characterized by insufficient GLUT4 expression, insulindysfunction (e.g., resistance, inactivity or deficiency) and/orinsufficient glucose transport into cells. Such diseases include, butare not limited to diabetes, hyperglycemia and obesity.

In another embodiment, the method of the invention can be used to createa novel in vitro model, in which GDF-8 is utilized to examine glucoseuptake or glucose metabolism in adipocytes. GDF-8, which is specificallyexpressed in muscle and fat in vivo, inhibits 3T3-L1 adipocytedifferentiation by directly or indirectly suppressing the expression ofadipocyte-specific genes, e.g. the GLUT4 transporter. GDF-8 can,therefore, be used as a prototype regulator of these genes in the 3T3-L1cell system. This system can be a model for understanding the role ofGDF-8 on the regulation of adipocyte-specific gene expression andprotein activity of molecules such as, but not limited to, transcriptionfactors, signal transduction proteins, leptin, fatty acid bindingprotein, fatty acid synthase, peroxisome proliferator-activatedreceptors, uncoupling proteins 1 and 2, and molecules that areactivated, inactivated, or modified by the actions of GDF-8.

Other uses for the methods of the invention will be apparent to one ofordinary skill in the art from the following Examples and Claims.

Administration of GDF-8 and GDF-11 Inhibitors in PharmaceuticalCompositions

GDF-8 and GDF-11 inhibitors used in the methods of the present inventionare generally administered to a subject in the form of a suitablepharmaceutical composition. Such compositions typically contain theinhibitor and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the GDF-8 inhibitor, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples ofsuitable routes of administration include parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethylenegrycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the GDF-8inhibitor in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the GDF-8 inhibitor into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the GDF-8inhibitor can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The GDF-8 inhibitor can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In one embodiment, the GDF-8 inhibitors are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.GDF-8 inhibitors which exhibit large therapeutic indices are preferred.While GDF-8 inhibitors that exhibit toxic side effects may be used, careshould be taken to design a delivery system that targets such GDF-8inhibitors to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any GDF-8inhibitor used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test GDF-8 inhibitor which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The GDF-8 inhibitors of the present invention, e.g., the anti-senseoligonucleotide inhibitors, can further be inserted into vectors andused in gene therapy. Gene therapy vectors can be delivered to a subjectby, for example, intravenous injection, local administration (see U.S.Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al.(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system

Vectors suitable for use in gene therapy are known in the art. Forexample, adenovirus-derived vectors can be used. The genome of anadenovirus can be manipulated such that it encodes and expresses a geneproduct of interest but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle. See for example Berkner etal. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitableadenoviral vectors derived from the adenovirus strain Ad type 5 dl324 orother strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known tothose skilled in the art. Recombinant adenoviruses can be advantageousin certain circumstances in that they are not capable of infectingnondividing cells. Furthermore, the virus particle is relatively stableand amenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use and thereforefavored by the present invention are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner etal., supra; and Graham et al. in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the gene of interest comprised in the nucleic acidmolecule can be under control of, for example, the E1A promoter, themajor late promoter (MLP) and associated leader sequences, the E3promoter, or exogenously added promoter sequences.

Yet another viral vector system useful for delivery of the GDF-8inhibitors of the invention is the adeno-associated virus (AAV).Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129). Adeno-associated viruses exhibit a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing asfew as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can beused to introduce DNA into T cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).Other viral vector systems that may be useful for delivery of the GDF-8inhibitors of the invention are derived from herpes virus, vacciniavirus, and several RNA viruses.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXEMPLIFICATION

Materials and Methods

The following studies were performed at Intracel (Rockville, Md.) usingsix week old male Balb/c mice. GDF-8 knockout and wild-type (control)mice were obtained from Dr. S. J. Lee (Johns Hopkins University, SeeMcPherron et al., Nature 387:83-90 (1997)).

Recombinant human GDF-8 was produced in Chinese Hamster Ovarian (CHO)cells. The secreted protein was purified using several steps ofchromatography to obtain substantially homogenous GDF-8.

Other materials and methods are described in the Examples below.

Example 1

Effect of GDF-8 Knockout on GLUT4 Protein Expression in Muscle Cells

To assess the impact on protein expression of the muscle cell glucosetransporter, GLUT4, of knocking out natural GDF-8 expression, samples ofvarious muscles were taken from both wild-type and GDF-8 knockout mice.Muscle samples were fixed in 10% (v/v) neutral buffered formaldehyde(StatLab, Lewisville, Tex.) for 8 hours at room temperature followed byembedding in Paraplast® X-tra tissue-embedding medium (Oxford Labware,St. Louis, Mo.). Cross sections of mouse muscle samples were prepared.Slides were preheated in an oven at 60° C. for at least 30 min prior toGLUT4 immunodetection. Paraffin sections were deparaffinzed in xylenethree times, 5 min each. Sections were rehydrated and then blocked with20% normal goat serum (Vector, Burlingame, Calif.) in “Antibody Diluent”(DAKO, Carpinteria, Calif.) for 20 minutes. Sections were incubated withrabbit anti-GLUT4 (Alpha Diagnostic International, San Antonio, Tex.)diluted in Antibody Diluent at a concentration of 2 μg/ml overnight atroom temperature. Sections were rinsed with OptiMax Wash Buffer(Biogenex, San Ramon, Calif.) and incubated with biotinylated goatanti-rabbit immunoglobulin (BioGenex) for conjugated streptavidin(BioGenex) for 30 min. Sections were rinsed with the wash buffer andthen DAB substrate (DAKO) was applied for visualizing the antibodybinding sites. Sections were counterstained with methyl green (DAKO) andmounted using Cytoseal™ 60 (Stephens Scientific, Riverdale, N.J.). Brownstaining indicates the expression of GLUT4 and green staining identifiesthe nucleus.

As shown in FIGS. 1A and 1B, GDF-8 knockout mouse samples displaysignificantly increased GLUT4 expression (as indicated by significantlyincreased staining with anti-GLUT4 antibody) compared to wild-typesamples, regardless of the type of muscle examined. This indicates thatGDF-8 causes a decrease in the expression of GLUT4 in these mice.

Example 2

Effect of GDF-8 Administration on GLUT4 Protein Expression in MuscleCells

The following study was performed to assess the converse of what wasfound in Example 1, i.e., whether exogenous GDF-8 represses theexpression of GLUT4 in muscle cells (as predicted from Example 1), andalso whether administration of GDF-8 can counteract the effects of theGLUT4 stimulator, insulin.

Mice were randomized to receive either an intramuscular (gastrcenemiusmuscle) injection of fifty microliters, containing 5 micrograms ofrecombinant human GDF-8 (in buffer containing 20 mM NaPO₄, 150 mM NaCl,0.1 mg/ml BSA, pH 6.5), or buffer alone. Twenty minutes later micereceived an intraperitoneal injection of either porcine insulinpurchased from Sigma Chemicals, St. Louis, Mo. (13 Units/kg in 0.1 ml ofthe same buffer used above but at pH 7.0), or buffer alone. One hourafter insulin administration the animals were sacrificed and samples ofthe injected muscle were removed.

As shown in FIG. 2, the administration of exogenous GDF-8 alone resultsin a significant decrease in GLUT4 expression in mouse gastrocnemiuscells. In contrast, the administration of insulin alone results in theopposite effect—a significant increase in GLUT4 expression (i.e.,staining) is observed in these cells. When GDF-8 and insulin aresimultaneously administered, the GLUT4 staining pattern appears close tothat of untreated control cells, suggesting that these two moleculeshave opposite regulatory effects on GLUT4.

Example 3

Effect of GDF-8-Expressing CHO Cell Tumors in Nude Mice

The results from the preceding Examples indicate that GDF-8 plays animportant role in the regulation of GLUT4 protein expression in musclecells. To further examine the role of GDF-8 in the regulation of overallglucose metabolism in vivo, a Chinese Hamster Ovarian (CHO) tumor cellline producing human GDF-8 (hGDF-8) was injected into nude mice to forma tumor expressing GDF-8. This CHO tumor cell injection approach hasbeen used as a model for determining the effects of various geneproducts in vivo (Black et al., Endocrinology 123:2657-2659 (1991)).

CHO cells expressing hGDF-8 were cultured in alpha medium with 0.1micromolar methotrexate and 1 mg/ml G418, while the control CHO cells(containing an empty expression vector) were cultured in alpha mediumwith 0.1 micromolar methotrexate. The cells were harvested bytrypsinization and resuspended in PBS at a concentration of 2×10⁷cells/ml. A subcutaneous injection of 1×10⁷ cells in 0.5 ml was madeinto the right thigh of male nu/nu NCR mice. Body weight and tumor sizeswere measured twice a week for the duration of the experiment. Northernblot analysis of mRNA isolated from the CHO GDF-8 tumors confirmed thatGDF-8 was expressed.

The systemic effects of the GDF-8 produced by the developing CHO GDF-8tumor were assessed. As shown in FIG. 3, the CHO tumors overexpressingGDF-8 caused dramatic total body weight loss (a decrease of 25%) within20 days in the nude mice, compared to their weight at the onset of theexperiment. In contrast, the mice harboring control CHO tumors notexpressing GDF-8 had a slight weight gain (FIG. 3).

As shown in FIG. 4, the CHO GDF-8 tumor-bearing mice showed an even moredramatic weight loss (35%) when the net body weights (total-tumor) werecompared with that of control tumor-bearing mice (FIG. 4, Panel A). Theweight loss was not due to the size of the CHO GDF-8 tumor, sincecontrol tumor weight was actually heavier than CHO GDF-8 tumor (FIG. 4,Panel B, and FIG. 5).

Individual tissues from CHO and CHO GDF-8-expressing tumor-bearinganimals were also isolated and weighed. Muscles and fat pads from CHOGDF-8 tumor-bearing animals showed a significant decrease in weightcompared to CHO tumor-bearing animals (FIG. 4, Panels C,D, and E). Thisgeneral wasting and reduction in skeletal muscle mass demonstrates thatthe GDF-8 protein produced from implanted CHO cells acts in a mannerstrictly compatible with, and as expected from, the results of the GDF-8knock-out approach.

To assess whether GDF-8 is involved in systemic glucose handling,wild-type nude mice carrying CHO-GDF-8 tumors were tested for elevatedglucose. As shown in FIG. 6, when compared to control CHO tumor-bearingmice, CHO GDF-8 tumor-bearing animals exhibited hyperglycemia and asignificant decrease in GLUT4 levels in muscle tissues. Taken togetherwith the fact that the GDF-8 knockout mice were hypoglycemic and hadincreased GLUT4 expression levels in muscle, these results suggestedthat GDF-8 increases glucose levels in the serum by inhibiting GLUT4levels in vivo.

Example 4

Systemic Effects of GDF-8 Knockout in Mice

Transgenic mice in which the GDF-8 gene is knocked out hadcharacteristic systemic problems, particularly hypoglycemia, significantmuscle hypertrophy, and a dramatic decrease in overall body fat. Thesefindings indicate not only that the modulation of GDF-8 may enable theregulation of glucose levels in the serum, thus serving as a treatmentfor diabetes, but also that GDF-8 may be useful in treating obesity andother disorders related thereto.

While GDF-8 knock-out mice provide a model for postulating the generalrole of GDF-8 in regulating muscle and fat growth and metabolicfunction, it is unclear whether the observed changes are a consequenceof embryonic GDF-8 deficiency or the result of post-natal development.Thus, this Example, as well as the immediately preceding Example (i.e.,Example 3 ) demonstrate for the first time that GDF-8 has an importantphysiological role in the adult animal. These two examples provideunambiguous support to the concept that modulating GDF-8 expression andactivity post-natally is a means of regulating muscle and fat growth andmetabolic function including, but not limited to, muscle growth, glucosehomeostasis and diabetes susceptibility.

Example 5

Effect of GDF-8 on the Differentiation of 3T3-L1 Pre-Adipocytes

To better characterize the effects of GDF-8 on glucose homeostasis,3T3-L1 cells were utilized as a model for adipocytes, a cell typeacutely responsive to insulin through its ability to increase hexosetransport through GLUT4. These cells have been well characterized as anexcellent model for adipogenesis (Hwang et al., Annu. Rev. Cell Dev.Biol. 13, 231-259 (1997), and MacDougald and Lane, Annu. Rev. Biochem.64, 345-373 (1995)). When these cells are stimulated with insulin,dexamethasone and isobutylmethylxanthine (IBMX), they are induced toundergo both morphological and biochemical changes resulting in theirdifferentiation into adipocytes.

When undifferentiated pre-adipocytes reached confluence, differentiationwas predictably achieved (Spiegelman et al., J. Biol. Chem. 268:6823-6826 (1993)) by successive replacements of their serum-containingDMEM media as follows: DMEM+serum+IBMX+dexamethasone+insulin for 2 days,DMEM+serum+insulin for 2 additional days. After this, the media wasagain replaced with DMEM+/−serum. GDF-8 and other growth factors wereadded at the onset of differentiation and were re-supplied at eachadditional medium change. Adipocytes were maintained for an additional 3to 5 days in this media for full differentiation to take place.

As shown in FIG. 7, GDF-8 inhibited differentiation of these 3T3-L1pre-adipocytes to adipocyte cells. The addition of GDF-8 to 3T3-L1 cellsat the onset of induction to differentiate into adipocytes prevented theconversion of pre-adipocytes to adipocytes, as seen by the maintenanceof pre-adipocyte morphology and the near-absence of refractile cellsthat contain lipid droplets (FIG. 7). In addition, as shown in FIG. 8,at the RNA level, GDF-8 inhibited the expression of GLUT4 mRNA, a knownadipocyte marker.

FIG. 7 also shows that GDF-8 is able to mimic the effects of both TNF-αand TGF-β₁ on GLUT4 mRNA levels. Importantly, GLUT4 is known to be thekey molecule responsible for insulin-sensitive glucose transport notonly in muscle tissue, but also in fat cells. Thus GDF-8 plays a role inmediating insulin resistance associated with Type II diabetes. GDF-8,which is specifically expressed in the muscle and in fat, can fullymimic the previously established effects of two broadly-expressedcytokines, namely TNF-α and TGF-β, on adipocyte differentiation andmetabolism (Szalkowski et al., Endocrinology 136: 1474-1481 (1995)). Dueto its specific expression, GDF-8 may be, among the three, thephysiologically most relevant polypeptide that regulates such processesin vivo.

Example 6

Effect of GDF-8 on Glucose Uptake in 3T3-L1 Adipocytes

In differentiated adipocytes, insulin stimulates glucose transportthrough the GLUT4 transporter in a dose-dependent fashion. Thus, theability of GDF-8 to interfere with this insulin-dependent glucose uptakemechanism was examined as follows.

Upon completion of differentiation, a glucose transport assay wasperformed on 3T3-L1 cells. GDF-8 was added in the final 72 hours ofdifferentiation. Insulin was added in Krebs-Ringer solution for 20 min.,followed by addition of [³H]-deoxyglucose (1 mCi/ml) for 10 min. Afterextensive washing, cells were lysed with Triton X-100 and thecell-associated radioactive glucose (due to GLUT4-mediated uptake) wasdetermined by scintillation counting.

As shown in FIG. 9, GDF-8 reduced the insulin-sensitivity of thesecells, as measured by fold-induction of glucose uptake, in adose-dependent manner. This reduced insulin-sensitivity of glucosetransport correlated with the decrease of GLUT4 mRNA levels in thesecells after GDF-8 treatment (FIG. 8). Thus, this assay offers an invitro correlate of GDF-8 activity that may be relevant to its in vivoeffects on body fat and muscle metabolic functions.

Besides a decrease in the GLUT4 mRNA levels (seen in FIG. 8), anadditional important observation was made that in the 3T3-L1 cells,GDF-8 actually increased basal glucose transport by about 50%. Thisincrease in baseline should also contribute to the reducedfold-increases in glucose uptake in response to insulin. Since thisbasal transport is mainly effected by the ubiquitous GLUT1 transporter(another hexose transporter), it indicates that the insulininsensitivity observed after GDF-8 treatment in adipocytes can stem froma combination of an increase in basal transport (through GLUT1 and otherglucose transporters) and a concomitant decrease in insulin-stimulatedtransport (through GLUT4). However, the increase in basal level ofglucose is not limited to the effect of GLUT1. Additional glucosetransporters can also increase the basal level of glucose in the 3T3-L1cells.

Example 7

Effect of GDF-8 in Diabetes Disease Models

The foregoing Examples demonstrate that GDF-8 inhibition can increaseGLUT4 transcription and expression, and thereby restore insulinsensitivity and reduce systemic glucose levels in a subject. Theforegoing Examples further demonstrate that GDF-8 inhibition upregulatesdifferentiation of adipocytes, and thereby increases insulin-sensitiveglucose uptake.

Together, this data suggests that interfering with GDF-8 function couldhave important applications for the treatment of Type II diabetes,obesity and disorders related to obesity. To pursue these potentialapplications, the following approaches can be taken.

A. Analysis of the Effect of the GDF-8 Mutation in Mouse Models ofObesity/Diabetes

GDF-8 knockout mice can be crossed with various mouse strains exhibitingobesity, particularly ob/ob, db/db, and mice carrying the lethal yellowmutation. Serum levels of known molecular markers of obesity, such asglucose, insulin, lipids, and creatine kinase are monitored and comparedto control animals lacking the GDF-8 knockout, as an indication of thepresence of this condition in the test animals. Functional assays fordiabetes/obesity including, but not limited to, an insulin sensitivityassay, a glucose tolerance assay and an ex-vivo glucose uptake byisolated muscle assay also can be performed to monitor the effect ofGDF-8 knockout on progeny mice.

In progeny mice carrying both the GDF-8 knockout and the recessiveobesity genotype, the lack of GDF-8 should suppress the obesityphenotype as compared to that of corresponding control mice, as measuredby serum levels of known molecular markers of obesity, such as glucose,insulin, lipids and creatine kinase. Additionally, the development ofdiabetes in these animals should be delayed or prevented by the absenceof GDF-8.

B. Analysis of GDF-8 Knockout Mice in Various Models of Diabetes

GDF-8 knockout mice can be subjected to agents capable of inducingexperimental diabetes, such as streptozotocin. An analysis of serumlevels of molecular diabetes markers, such as glucose, insulin, lipids,and creatine kinase is performed in these animals, and compared to,e.g., streptozotocin-treated wild-type control animals. Functionalassays for diabetes/obesity including, but limited to, an insulinsensitivity assay, a glucose tolerance assay and an ex-vivo glucoseuptake by isolated muscle assay can be performed to monitor the effectof streptozotocin on the treated and non-treated animals. The GDF-8knockout mice should be relatively resistant to such treatments, and theonset of experimental diabetes should be altogether prevented, delayed,or be less severe.

C. Demonstration of the Efficacy of GDF-8 Inhibitors in Mouse Models ofObesity/Diabetes

Mice serving as models for obesity or diabetes can be treated with GDF-8inhibitors to determine whether inhibition of GDF-8 and thecorresponding impact on GLUT4 ameliorates the symptoms of either obesityor diabetes in these animals.

Mice with either obesity or diabetes are treated with one or more GDF-8inhibitors in a therapeutically effective dose. GDF-8 levels in treatedand control mice can be assessed by Western blot analysis usingantibodies specific for GDF-8. Levels of molecules characteristic forobesity and diabetes, such as glucose, insulin, lipids, and creatinekinase can be assessed in serum samples taken from treated and controlanimals. Functional assays for diabetes/obesity including, but notlimited to, an insulin sensitivity assay, a glucose tolerance assay andan ex-vivo glucose uptake by isolated muscle cell assay can be performedto monitor the effect of the inhibitor on treated and non-treatedanimals. Similarly, muscle and fat cell differentiation can be observedin these animals. Analysis of such studies should enable a determinationof the overall effect of the inhibition of GDF-8 on the progression ofdiabetes or obesity in animal models for these diseases.

All patents, published patent applications and other publishedreferences disclosed herein are hereby expressly incorporated herein intheir entireties by reference.

Equivalents

Those skilled in the art will recognize, or will be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1. A method of increasing expression of GLUT4 in a subject comprising administering to the subject a GDF-8 inhibitor, wherein the GDF-8 inhibitor is selected from the group consisting of a peptide fragment of GDF-8, a dominant-negative mutant of GDF-8, a GDF-8 receptor antagonist, an antisense nucleic acid which binds GDF-8 mRNA, an antibody or antibody fragment which binds to GDF-8 or its receptor, and an anti-GDF-8 ribozyme.
 2. A method of increasing insulin sensitivity and glucose uptake by cells in a subject comprising administering to the subject a GDF-8 inhibitor wherein the GDF-8 inhibitor is selected from the group consisting of a peptide fragment of GDF-8, a dominant-negative mutant of GDF-8, a GDF-8 receptor antagonist, an antisense nucleic acid which binds GDF-8 mRNA, an antibody or antibody fragment which binds to GDF-8 or its receptor, and an anti-GDF-8 ribozyme.
 3. A method of treating diabetes in a subject comprising administering to the subject a GDF-8 inhibitor wherein the GDF-8 inhibitor is selected from the group consisting of a peptide fragment of GDF-8, a dominant-negative mutant of GDF-8, a GDF-8 receptor antagonist, an antisense nucleic acid which binds GDF-8 mRNA, an antibody or antibody fragment which binds to GDF-8 or its receptor, and an anti-GDF-8 ribozyme.
 4. The method of any one of claims 1-3, wherein the GDF-8 inhibitor is an antibody or antibody fragment.
 5. (canceled)
 6. The method of any one of claims 1-3, wherein the peptide fragment of GDF-8 is derived from mature GDF-8 protein.
 7. The method of any one of claims 1-3, wherein the peptide fragment of GDF-8 is derived from the pro domain of a GDF-8 protein.
 8. The method of claim 2, wherein said insulin sensitivity and glucose uptake is increased by modulating the expression of a hexose transporter selected from the group consisting of GLUT4 and GLUT1.
 9. The method of claim 2, wherein the cell is a muscle cell or a precursor thereof.
 10. The method of claim 2, wherein the cell is an adipocyte or a precursor thereof.
 11. The method of claim 3, wherein the subject is suffering from type II diabetes.
 12. The GDF-8 inhibitor of claim 7, wherein the inhibitor comprises amino acids 1-266 of the pro-domain.
 13. The GDF-8 inhibitor of claim 1, wherein the peptide fragment of GDF-8 spans the C-terminus of the mature GDF-8. 